The development of immunoassays for the detection of antibodies has been limited by difficulties in producing sufficient quantities of specific antigens that are essentially free of immunoreactive contaminants. The presence of contaminants that react with antibodies present in patient samples results in lower assay specificity and sensitivity and an increase in false positive results. The production of large amounts of antigen enables easier purification of antigen having a higher degree of purity and thus less immunoreactive contaminants.
The present invention overcomes the difficulties by providing a simple and highly efficient expression system that allows for the production of large quantities of antigens. The invention relies on the efficient expression resulting from the inclusion of the nucleotide sequence AGGAGGGTTTTTCAT (which corresponds to nucleotides 1-15 of SEQ ID NO.:1) directly upstream from the ATG codon which marks the start of translation.
The invention is particularly useful for the expression of viral antigens of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV).
HIV is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). The nucleic acid sequence of the HIV proviral genome has been deduced and the location of various protein coding regions within the viral genome has been determined. Of particular interest to the present invention are the portions of the HIV genome known in the art as the gag and env regions. The gag region encodes a precursor protein that is cleaved and processed into three mature proteins, p17, p24 and p15. The HIV p24 protein has an apparent relative molecular weight of about 24,000 daltons and is known in the art as the HIV core antigen because it forms the viral capsid. Also of interest is the env region which encodes the envelope glycoproteins gp120 and gp41, which are required forviral entry into the cell. The first step in infection is the formation of a complex of gp120, gp41 and the cellular CD4 protein, binding the virus particle to the cell. The formation of this complex appears to alter the conformation of gp41, allowing its interaction with a second cellular protein “fusin”, an interaction required for HIV entry into the cell.
The p24 antigen of HIV is of particular interest because studies have indicated that the first evidence of anti-HIV antibody formation (sero-conversion) in infected individuals is the appearance of antibodies induced by the p24 antigen, i.e., anti-p24 antibodies. In addition, recent studies have reported that p24 protein can be detected in blood samples even before the detection of anti p24 antibodies. Detecting the presence of either the p24 protein or anti-p24 antibodies therefore appears to be the best approach to detecting HIV infection at the earliest point in time. Furthermore, the p24 antigen reappears in the blood of infected individuals concomitant with the decline of anti-p24 antibody in patients showing the deterioration in their clinical condition that accompanies transition into full-blown AIDS. Thus, the p24 antigen can serve as an effective prognostic marker in patients undergoing therapy.
Most cases of Non-A, non-B hepatitis (NANBH) are caused by the transmissible virus now designated as hepatitis C virus (HCV). Isolates of HCV nucleic acids have been obtained and completely characterized at the sequence level. The HCV genome is comprised of a plus strand RNA molecule that codes for a single polyprotein which is cleaved to produce functionally distinct structural and nonstructural HCV proteins. Structural proteins include the capsid and envelope proteins which form the viral particle. Nonstructural proteins, such as helicase and RNA-directed RNA polymerase are required for viral function.
Some HCV gene products, or portions thereof have been expressed as fusion products. The HCV antigen C-100-3, derived from portions of the nonstructural genes designated NS3 and NS4, has been expressed as a fusion protein and used to detect anti-C-100-3 antibodies in patients with various forms of NANB hepatitis. See, for example, Kuo et al, Science, 244:362-364 (1989) and International Application No. PCT/US88/04125. A diagnostic assay based on C-100-3 antigen is commercially available from Ortho Diagnostics, Inc. (Raritan, N.J.). However, the C-100-3 antigen-based immunoassay has been reported to preferentially detect antibodies in sera from chronically infected patients. C-100-3 seroconversion generally occurs from four to six months after the onset of hepatitis, and in some cases C-100-3 fails to detect any antibody where an NANBV infection is present. Alter et al, New Eng. J. Med., 321:1538-39 (1989); Alter et al, New Eng. J. Med., 321:1494-1500 (1989); and Weiner et al, Lancet, 335:1-3 (1990). McFarlane et al, Lancet, 335:754-757 (1990), described false positive results when the C-100-3-based immunoassay was used to measure antibodies in patients with autoimmune chronic active hepatitis. In addition, Grey et al., Lancet, 335:609-610 (1990), describe false positive results using C-100-3-based immunoassay on sera from patients with liver disease caused by a variety of conditions other than HCV. Houghton et al., U.S. Pat. No. 5,350,671, have disclosed a series of fusion proteins which include amino acids from parts of various structural and nonstructural HCV gene products fused to superoxide dismutase (SOD), many of which have no immunogenic activity when tested against HCV positive antisera.
A NANBV immunoassay that could accurately detect seroconversion at early times after infection, or that could identify an acute NANBV infection, is not presently available.
The present invention provides compositions of recombinantly produced HIV and HCV antigens, free of bacterial and other viral components, thus enabling the detection of HIV and HCV antibodies with improved accuracy and sensitivity. The present invention also enables high yield expression of these antigens alone or as fusion proteins.